Characteristic hexasaccharide sequences in octasaccharides derived from shark cartilage chondroitin sulfate D with a neurite outgrowth promoting activity.
A mouse brain chondroitin sulfate (CS) proteoglycan, DSD-1-PG, bears the DSD-1 epitope and has neurite outgrowth promoting properties. Shark cartilage CS-C inhibits the interactions between the DSD-1-specific monoclonal antibody 473HD and the CS chains of the DSD-1-PG, which is expressed on the mouse glial cells (Faissner, A., Clement, A., Lochter, A., Streit, A., Mandl, C., and Schachner, M. (1994) J. Cell Biol. 126, 783-799). On the other hand, several hexasaccharides isolated from commercial shark cartilage CS-D, which contains a higher proportion of characteristic D units (GlcUA(2-sulfate)beta1-3GalNAc(6-sulfate)) as compared with CS-C, has the A-D tetrasaccharide sequence composed of an A disaccharide unit (GlcUAbeta1-3GalNAc(4-sulfate)) and a D disaccharide unit (Nadanaka, S. and Sugahara, K. (1997) Glycobiology 7, 253-263). In this study, the biological activities and the structure of shark cartilage CS-D were investigated. CS-D inhibited the interactions between monoclonal antibody 473HD and DSD-1-PG and also promoted neurite outgrowth of embryonic day 18 hippocampal neurons. Eight octasaccharide fractions were isolated from CS-D after partial digestion with bacterial chondroitinase ABC by means of gel filtration chromatography and anion-exchange high performance liquid chromotography to investigate the frequency and the arrangement of the A-D tetrasaccharide unit in the polymer sequence. Structural analysis performed by a combination of enzymatic digestions with 500-MHz 1H NMR spectroscopy demonstrated that the isolated octasaccharides shared the common core structure DeltaHexAalpha1-3GalNAcbeta1-4(GlcUAbeta1-3GalNAc)3 with four, five, and six sulfate esters at various hydroxyl groups in different combinations. In the structure, DeltaHexA and GlcUA represent 4-deoxy-alpha-L-threo-hex-4-enepyranosyluronic acid and glucuronic acid, respectively. No D-D tetrasaccharide sequence was found, and discrete D disaccharide units were demonstrated exclusively as A-D tetrasaccharide units in either an A-D-A or an A-D-C hexasaccharide sequence in the five octasaccharides that represented about 5.0% (w/w) of the starting polysaccharides (C denotes the disaccharide GlcUAbeta1-3GalNAc(6-sulfate)). It remains to be determined whether such characteristic hexasaccharide sequences present in shark cartilage CS-D serve as functional domain structures recognized by some protein ligands.
Nadanaka
S
S
Department of Biochemistry, Kobe Pharmaceutical University, Higashinada-ku, Kobe 658, Japan.
Clement
A
A
Masayama
K
K
Faissner
A
A
Sugahara
K
K
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D019765:Chondroitin ABC Lyase / Q000378:metabolism; D002809:Chondroitin Sulfates / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D008969:Molecular Sequence Data; D016501:Neurites / Q000187:drug effects*; D009844:Oligosaccharides / Q000737:chemistry* / Q000494:pharmacology; D012754:Sharks; D013379:Substrate Specificity
D000911:Antibodies, Monoclonal; D009844:Oligosaccharides; C065068:chondroitin sulfate D; D002809:Chondroitin Sulfates; D019765:Chondroitin ABC Lyase